This proposal describes genetic and biochemical analyses of the replication of the Moloney murine leukemia virus, the prototype of the simple mammalian retroviruses. We are broadly interested in all aspects of the viral life cycle, but are especially focused on identifying and characterizing the cellular host factors that interact with the virus. In this proposal we will address three host systems that act to limit or restrict virus replication. First, we will characterize the mechanisms by which embryonic stem (ES) cells transcriptionally silence proviral DNAs and maintain the integrity of the germ line. We will study two parallel pathways - a rapid and efficient mechanism targeting a specific DNA element of the Moloney provirus, and a slower, less efficient one acting more broadly on many proviruses -- and determine how these silencing mechanisms are specifically active in ES cells. These experiments will provide important new information about the properties that define stemness - the pluripotent state of ES cells. Second, we will explore the role of SUMOylation, the covalent addition of SUMO (small ubiquitin-related modifier) to particular protein substrates, in virus replication and restriction. We will test for SUMOylation of the viral capsid protein during infection, and explore the potential role of SUMOylation in the capsid-dependent restriction of virus infection by the host genes Fv1 and TRIM5-alpha. Third, we will examine the mechanism of expression of the Gag-Pol precursor protein, which requires the suppression of translational termination at a UAG stop codon at the 3' end of the gag gene. This readthrough event depends on an RNA pseudoknot immediately downstream of the stop codon. We will identify new regulators of readthrough by isolation of RNA-protein complexes from infected cells. Finally, we will explore the mechanisms by which the MuLV mRNA for Gag and Gag-Pol evades the Nonsense-Mediated mRNA Decay (NMD) system. We will test the hypothesis that an interaction between the retroviral reverse transcriptase and the host translational termination factor eRF1 prevents normal recognition of the viral mRNA by NMD and protects them from degradation. All three areas of investigation will advance our understanding of host defense systems and may provide important new targets for antiviral intervention. Most importantly, these experiments will significantly extend our understanding of fundamental aspects of retrovirus replication, and of new cell biological processes that impact on these important viruses.